Podocalyxin Regulates Murine Lung Vascular Permeability by Altering Endothelial Cell Adhesion
Figure 6
Podocalyxin deletion results in increased vascular permeability without altered endothelial cell frequency or immunophenotype.
(A) Vascular permeability in lungs assessed by a modified Miles assay. Female mice were treated with PBS (naïve) or LPS (2 mg/kg) intra-tracheally 24 h before sacrifice PodxlF/F (black bars), and PodxlΔEC (white bars) (n = 5–7 mice per genotype). One hour before sacrifice, mice received 20 mg/kg Evan's blue dye via the lateral tail vein. *Significantly different with P<0.05 by when compared to PodxlF/F naïve mice. #Significantly different (P<0.05) than LPS treated PodxlF/F mice by one-way ANOVA. (B) Lung edema presented as a ratio of wet/dry lung weight. Lungs were excised and weighed immediately for wet weight and subsequently dried at 60°C for 36 h and weighed again to determine the dry weight (n = 6 mice). (C) Vessel density was determined by the ratio of von Willebrand factor-positive (vWF+) staining to total lung tissue. Data represent 4 images per mouse and 6 mice per group. (D) Lung tissue displays normal expression of endothelial cell markers by flow cytometry. Shown are representative flow cytometry histograms. The frequency of CD31+ endothelial cells from each genotype is shown in the first profile. All subsequent profiles were gated on CD31+ cells in order to focus exclusively on marker expression by endothelia. (E) Expression of junctional proteins in lung. Lungs from PodxlF/F and PodxlΔEC mice were homogenized in RIPA buffer and proteins were resolved on 8% SDS-PAGE gel followed by immunoblotting with the antibodies indicated. Actin was used as an internal loading control. The immunoblots shown are from one experiment and are representative examples of 4 independent mice/genotype. (F) Localization of junctional proteins in lung. Sections from inflated lungs of PodxlF/F and PodxlΔEC mice were stained for ZO-1 (green), claudin-5 (red), and nuclei (DAPI, blue). In endothelial cells, ZO-1 and claudin-5 co-localize (yellow), and ZO-1 is also detected in epithelial cell junctions (green). The immunofluorescence micrographs are representative of 3 images per mouse and 3 mice per genotype.